SUMMARY

Exposure of marine invertebrates to high temperatures leads to a switch
from aerobic to anaerobic metabolism, a drop in the cellular ATP concentration
([ATP]), and subsequent death. In mammals, AMP-activated protein kinase (AMPK)
is a major regulator of cellular [ATP] and activates ATP-producing pathways,
while inhibiting ATP-consuming pathways. We hypothesized that temperature
stress in marine invertebrates activates AMPK to provide adequate
concentrations of ATP at increased but sublethal temperatures and that AMPK
consequently can serve as a stress indicator (similar to heat shock proteins,
HSPs). We tested these hypotheses through two experiments with the rock crab,
Cancer irroratus. First, crabs were exposed to a progressive
temperature increase (6°C h–1) from 12 to 30°C. AMPK
activity, total AMPK protein and HSP70 levels, reaction time, heart rate and
lactate accumulation were measured in hearts at 2°C increments. AMPK
activity remained constant between 12 and 18°C, but increased up to
9.1(±1.5)-fold between 18 and 30°C. The crabs' reaction time also
decreased above 18°C. By contrast, HSP70 (total and inducible) and total
AMPK protein expression levels did not vary significantly over this
temperature range. Second, crabs were exposed for up to 6 h to the sublethal
temperature of 26°C. This prolonged exposure led to a constant elevation
of AMPK activity and levels of HSP70 mRNA. AMPK mRNA continuously increased,
indicating an additional response in gene expression. We conclude that AMPK is
an earlier indicator of temperature stress in rock crabs than HSP70,
especially during the initial response to high temperatures. We discuss the
temperature-dependent increase in AMPK activity in the context of Shelford's
law of tolerance. Specifically, we describe AMPK activity as a cellular marker
that indicates a thermal threshold, called the pejus temperature,
Tp. At Tp the animals leave their
optimum range and enter a temperature range with a limited aerobic scope for
exercise. This Tp is reached periodically during annual
temperature fluctuations and has higher biological significance than earlier
described critical temperatures, at which the animals switch to anaerobic
metabolism and HSP expression is induced.

INTRODUCTION

Marine crustaceans are exposed to frequent seasonal and diel temperature
changes. These temperature changes have a profound impact on ectothermal
animals' energy metabolism and scope for exercise. Additionally, the potential
impacts of global climate change on marine ecosystems emphasize the need to
understand the effects of rising sea temperatures on marine invertebrates (for
a review, see Osovitz and Hofmann,
2007). To predict how species will be affected by these
temperature changes, we need both an indicator of heat stress and an
understanding of the underlying mechanisms. Many physiological studies
investigating temperature stress identify heat stress through the expression
of heat shock proteins (HSPs) (e.g. Feder
and Hofmann, 1999; Hoffmann et
al., 2003; Tomanek,
2005). HSPs are ubiquitously expressed proteins that act as
chaperones and aid in the conformational stabilization of other proteins
(Feige et al., 1996;
Frydman, 2001). Though HSPs
were identified and named for their activation during temperature stress, many
other stressors, including osmotic shock, hypoxia and exercise, induce HSP
expression (for a review, see Hochachka
and Somero, 2002). Named after their molecular weight, the HSP70
family includes constitutive (or cognate) HSP73, and stress-inducible HSP72
(Reading et al., 1989;
Brown et al., 1993). Tomanek
and Somero have shown that a heat shock response in snails is already
detectable 1–2 h after heat stress and that the temperature at which
HSP70 expression occurs can change after acclimation
(Tomanek and Somero, 1999).
Iwama acknowledges that HSPs are good indicators of general cellular stress,
but points out that due to the variety of possible triggers of HSP expression,
specific conclusions about HSP up- or down-regulation may not be valid
(Iwama et al., 2004). The time
lag between the actual heat stress and the detectable accumulation of HSPs
raises the question of whether an additional, faster mechanism exists that
would aid in withstanding thermal challenges.

In mammals, AMP-activated protein kinase (AMPK) has been described as a
`low fuel sensor' (Hardie and Carling,
1997) or a `metabolic master switch'
(Winder and Hardie, 1999),
which reflects AMPK's central role in cellular energy metabolism. AMPK is
thought to constantly monitor the energy status of the cell and, if needed,
regulate the anabolic and catabolic processes to ensure a constant ATP
concentration (Hardie et al.,
2006). This regulation is achieved by AMPK through phosphorylation
and subsequent activation or inhibition of rate-limiting enzymes of all the
major energy metabolism pathways. By decelerating ATP-consuming pathways such
as glycogen synthesis, fatty acid synthesis, protein synthesis and cholesterol
synthesis, and in parallel accelerating ATP-producing pathways such as glucose
uptake, glycolysis and fatty acid β-oxidation, the activation of AMPK
prevents ATP depletion and promotes replenishment of the ATP pool
(Fig. 1) (for a review, see
Hardie et al., 2006;
Hardie and Sakamoto,
2006).

Model of the AMP-activated protein kinase (AMPK) cascade. Stressors such as
hypoxia, exercise or temperature lead to a decrease in cellular ATP and an
increase in cellular AMP. This activates AMPK either directly, or indirectly
through an upstream AMPK kinase. Once AMPK is activated, it phosphorylates
multiple downstream targets, mainly rate-limiting enzymes of all energy
metabolism pathways. The effect of this phosphorylation, in summary, leads to
an acceleration of all ATP-producing pathways and a deceleration of all
ATP-consuming pathways. Therefore, AMPK activation preserves the cellular ATP
concentration.

The AMPK protein is a heterotrimer with a catalytic α-subunit and
regulatory β- and γ-subunits. AMPK derives its main activation by
phosphorylation of the α-subunit at threonine 172 (T172). AMP plays a
major role in activating AMPK by four effects in parallel: (1) allosteric
activation of AMPK kinase (AMPKK); (2) binding of AMP to AMPK, rendering it a
poorer substrate for protein phosphatases; (3) binding of AMP to AMPK, making
it a better substrate for the upstream kinase, AMPKK; and (4) allosteric
activation of AMPK (Hardie et al.,
1999). AMP is a good indicator of cellular stress because an
increased ATP hydrolysis rate leads to a rapid accumulation of AMP in the
cell. ATP hydrolysis first increases the cellular ADP concentration. The ADP
is then converted by the adenylate kinase reaction (2 ADP→ATP+AMP) to ATP
and AMP (Hardie et al., 2003).
Therefore, during increased ATP use, AMP accumulates well before any changes
in cellular ATP or ADP concentration occur. This is especially true for muscle
tissues with the creatine phosphate or arginine phosphate system. These high
energy phosphagens rapidly provide more ATP, so that the cellular ATP
concentration remains constant despite a high ATP hydrolysis rate
(Bessman, 1985). In a previous
study on mice, we described AMPK activation under conditions of a raised AMP
but constant ATP concentration (Frederich
and Balschi, 2002).

Hypoxia, exercise and osmotic shock are known to activate AMPK in mammals
through AMPKK activation and AMP accumulation. Cold stress has been shown to
affect AMPK activity in frogs (Bartrons et
al., 2004) and in the brown adipose tissue of mice
(Mulligan et al., 2007); for a
recent review on AMPK-activating factors see Hardie et al.
(Hardie et al., 2006).
However, AMPK activity and its regulation during heat stress, especially in
invertebrates, have not yet been investigated thoroughly. Furthermore, most
AMPK studies focus on vertebrates, especially mammals. Only a few studies have
investigated AMPK in invertebrates in species such as the brine shrimp,
Artemia franciscana (Zhu et al.,
2007), and the fruit fly, Drosophila melanogaster
(Lee et al., 2007).

AMPK is remarkably highly conserved during evolution with high sequence
similarity between humans and other mammals [rat, mouse, rabbit, pig
(Hardie et al., 1998)]. AMPK
has also been described in the fruit fly, D. melanogaster
(Pan and Hardie, 2002), and
the nematode worm, C. elegans
(Gao et al., 1995), as well as
in plants such as cauliflower and tobacco
(Kelner et al., 2004). We
recently identified AMPK in the rock crab, Cancer irroratus, and
demonstrated tissue-specific AMPK activation during hypoxia
(Pinz et al., 2005). It is
therefore likely that the AMPK cascade is a central mechanism for regulating
energy metabolism found in most, if not all eukaryotes.

Marine invertebrates switch to anaerobiosis during heat stress (see
Discussion). This anaerobic metabolism is characterized by a limited ATP
yield, and an accumulation of AMP is expected due to the concomitant increase
in metabolic rate. Because of the ubiquity of the AMPK cascade, we predicted
that AMPK is activated during temperature stress. We tested this hypothesis in
a marine decapod crustacean and compared AMPK activation with a more
established marker for heat stress, the heat shock protein 70 (HSP70).

MATERIALS AND METHODS

Animals

Male rock crabs, Cancer irroratus (Say 1817), with an average
carapace width of 101.9±9.7 mm (mean±s.d.) were obtained from a
local lobster fisherman in Saco, ME, USA, kept in a flow-through seawater
system in the Marine Science Center of the University of New England at
12–15°C, and fed squid and fish ad libitum.

Temperature incubations

Animals (five per experiment) were placed in a darkened 100 l tank at
12°C overnight. The next day animals were exposed to a fast progressive
temperature increase (6°C h–1) and killed at 12, 16, 18,
20, 22, 24, 26 or 28°C (30°C was also used in some trials). Animals
were killed at the respective temperature with a cut through the cerebral
ganglion and the heart was removed and either stored in RNAlater® solution
(Ambion, Austin, TX, USA) or flash-frozen with Wollenberger tongs pre-cooled
in liquid nitrogen. The flash frozen samples were stored at –80°C
until analysis of AMPK activity and HSP70 protein levels.

In a second set of experiments, animals were exposed to the same
progressive temperature increase up to the sublethal temperature of 26°C
and temperature was then kept constant at 26°C. Animals (five per time
point) were killed and tissue harvested as described above at 0, 1, 2, 4 and 6
h after reaching 26°C.

Reaction to experimental stimulation

To investigate the ability of the animals to respond to experimental
stimulation at different temperatures, animals were subjected to the fast
progressive temperature increase described above. At 12, 16, 18, 20, 22, 24,
26, 28 and 30°C the animals were turned upside down and placed on a flat
surface underwater. The time (reaction time) to return to the upright position
was monitored. Animals were counted as `not responding' if they did not turn
within 15 min.

Heart rate

To monitor the animals' heart rate during the temperature incubations,
photoplethysmographs (iSiTEC, Bremerhaven, Germany) connected to a digital
recording device (PowerLab, Mountain View, CA, USA) were glued to the carapace
above the heart as described in detail by Depledge
(Depledge, 1984) or Frederich
and Pörtner (Frederich and
Pörtner, 2000).

Lactate

The lactate concentration in the heart tissue was measured using a
photometric test according to Bergmeyer
(Bergmeyer, 1985) to
characterize the onset of anaerobiosis. Briefly, tissue was ground under
liquid nitrogen and the frozen tissue powder transferred to 1.2 mol
l–1 perchloric acid to precipitate the protein. After
neutralization with 1 mol l–1 K2HPO4,
the sample was centrifuged, and the lactate concentration was measured in the
supernatant at 340 nm as NADH accumulation by the lactate dehydrogenase
reaction. The lactate concentrations were normalized to the protein
concentration in the extract and are presented as nanomoles per gram of
protein (Bradford, 1976).

The AMPK activity, quantified as T172 phosphorylation, could also be
interpreted as AMPKK activity rather than AMPK activity itself. Because AMPK
has a multitude of downstream targets, the activity of each target (e.g.
acetyl-CoA carboxylase, HMG CoA reductase, phosphofructokinase 2, protein
synthase, and many others) could be used to measure AMPK activity. Each of
these downstream targets, however, is regulated in its respective activity by
other regulators as well and, therefore, does not reflect AMPK activity alone.
Many studies, including one of ours
(Frederich and Balschi, 2002),
have shown that an increase in phosphorylated T172 is representative of the
respective cellular effects of activated AMPK. These days, it is common to
quantify AMPK activity as T172 phosphorylation.

We measured the heat shock response as both the total HSP70 and the
inducible HSP70 to ensure that we did not miss any small upregulation of
inducible HSP that might otherwise be masked by a constitutively expressed HSP
in the cell.

Sequencing

To design degenerate primers, we searched GenBank for AMPKα and
AMPKγ protein sequences from various invertebrate and vertebrate
species. Obtained sequences were aligned using the MultAlin tool
(http://bioinfo.genotoul.fr/multalin/multalin.html).
Degenerate forward and reverse primers for PCR (see
Table 1) were designed based on
highly conserved areas in the aligned sequences. For HSP70, we used a primer
pair that Voznesensky and colleagues
(Voznesensky et al., 2004)
prepared for the copepod, Calanus finmarchicus.

Nucleotide sequence of Cancer irroratus primers used for
amplification of AMPK, HSP70 and 18S

Total RNA from C. irroratus hepatopancreas was purified using the
Total RNA Isolation System (Promega, Madison, WI, USA) and reverse transcribed
(Super-Script First Strand Synthesis System, Invitrogen, Carlsbad, CA, USA).
cDNA was amplified with the respective primer pairs via PCR with an
annealing temperature of 45°C. DNA was sequenced at the Mount Desert
Island Biological Laboratory (MDIBL, Salisbury Cove, ME, USA) sequencing core
facility on an ABI 3100 sequencer. The DNA sequences obtained were converted
to a predicted amino acid sequence using the NCBI open reading frame finder
(http://www.ncbi.nlm.nih.gov),
and a BLAST search confirmed the cDNA as AMPK or HSP70, respectively. These
methods are described in detail by Towle and colleagues
(Towle et al., 2001).

Quantitative real-time PCR

Specific primers for AMPK and HSP70 (see
Table 1) were designed with the
idtDNA primer design tool
(www.idtDNA.com)
based on the sequences obtained with the respective degenerate primers.
Specific primers for 18S were designed based on Spears et al.
(Spears et al., 1992) (GenBank
accession no. M91050). Expression of AMPK and HSP70 mRNA was quantified in
duplicate by quantitative real-time PCR using the Stratagene Brilliant SYBR
Green qPCR Kit (Stratagene, La Jolla, CA, USA) on a Stratagene MX3005s
instrument. After 40 cycles with an annealing temperature of 55°C, a
melting curve analysis confirmed that only one DNA product was amplified. The
18S gene was used as a reference gene and one sample (undiluted and diluted
1:10, 1:100, 1:1000) with high HSP70 mRNA expression served as an internal
standard. AMPK and HSP70 mRNA levels are shown as relative increase above the
AMPK or HSP70 mRNA level, respectively, for heart tissues at the control
temperature of 12°C.

Alignment of AMPKα amino acid sequences for vertebrate and
invertebrate species. The sequence for Cancer irroratus is from this
study. The remaining sequences were obtained from GenBank (Aedes
aegypti AAX20150, Artemia franciscana ABI13783, Rattus
norvegicus NM_023991). Sequence conservation is indicated as: black, no
conservation; blue, some conservation; and red, complete conservation among
the compared species. More than 60% of the obtained Cancer irroratus
sequence (453 amino acids) is conserved in this comparison. A large region of
conservation is found in the area flanking the T172 region that activates the
AMPK protein. T172 lines up in this sequence comparison at position 176
because it was identified and named in the rat sequence, but shifts slightly
when compared with other species.

Statistics

Data were tested for significant difference by ANOVA and a Tukey
post-hoc analysis or repeated measures ANOVA, dependent on the data
set (GraphPad InStat;
www.graphpad.com).
P<0.05 was considered significant. Data are shown as means±
s.e.m.

RESULTS

AMPK sequences are highly conserved

The partial amino acid sequence for the AMPKα subunit for C.
irroratus (GenBank submission no. FJ496868) confirmed the high
conservation that has been reported for several other species
(Hardie et al., 1998). More
than 60% of the obtained C. irroratus sequence (453 amino acids) is
conserved in comparison to other arthropods and vertebrates
(Fig. 2). We could not
determine whether the C. irroratus sequence more closely resembles
the mammalian AMPKα1 or α2 subunit based on our partial sequence.
However, more importantly, the region flanking the regulatory threonin 172
(T172) site where the AMPKα subunit is phosphorylated by an upstream
AMPKK is highly conserved. The region flanking the T172 position for C.
irroratus contains the amino acid sequence VDGEFL - RpTSCGSPNY,
compared with rat SDGEFLRpTSCGSPNY. The antibodies used to quantify
AMPK phosphorylation in C. irroratus (see below) were raised against
the rat specific antigen KDGEFLRpTSCGSPNY. Except for the very first
amino acid, rat and crab sequences are identical. The antigen used also varies
in this first position. Exactly the same Cancer irroratus sequence of
15 amino acids flanking the T172 position was identified for Carcinus
maenas, Homarus americanus and Calanus finmarchicus (data not
shown). With this high sequence conservation of the peptide, the use of
heterologous antibodies is not problematic. Similarly high conservation was
observed in the 180 amino acid sequence of the AMPKγ subunit (GenBank
submission no. FJ496867, 54% similar to mouse, NM_153745). As expected, the
obtained HSP70 sequence of 178 amino acids (GenBank submission no. FJ496866)
is highly conserved as well and shows more than 80% similarity with the mouse
HSP70 sequence (AAC84170).

Effect of progressive temperature increase

Reaction to experimental stimulation

Between 12 and 18°C, the crabs needed 4.1±0.9 s to right
themselves after being turned upside down
(Fig. 3A). The crabs became
slower at 20, 22 and 24°C (28.5, 58.9 and 46.2 s, respectively). With a
high variability (between 1 and 280 s) this increase was not statistically
significant. At 26°C, the average reaction time decreased to
20.3±9.8 s. However, 20% of the crabs did not return to the upright
position (Fig. 3B) and are not
included in this time average. At 28°C, 80% were not responding at all,
and the remaining animals needed 157.7±20.6 s to react
(P<0.05, repeated measures ANOVA). None of the animals were able
to right themselves at 30°C.

Heart rate

During the progressive temperature increase, the heart rate increased,
following a Q10 of 2.2 between 12 and 26°C, reaching a
maximum of 153±27 beats min–1 at 26°C. Heart rate
remained constant between 26 and 30°C, and then dropped at temperatures
above 30°C (Fig. 3C).

Lactate

Lactate concentration in the heart remained constant at 7.3±2.6 nmol
g–1 protein between 12 and 26°C, but increased
significantly (P<0.05, ANOVA) 2.2-fold to 16.1±5.6 nmol
g–1 protein above 26°C
(Fig. 3D). The concordance
between the lack of scope for exercise, the maximum heart rate (see above) and
an increase in lactate accumulation indicates that the animals reached their
critical temperature (Tc, see Discussion) between 26 and
28°C, as indicated by the dashed line in
Fig. 3. However, a reduction in
the scope for exercise is already evident between 18 and 20°C, indicated
by the dotted line in Fig. 3
(Tp).

(A) Reaction time after experimental stimulation in Cancer
irroratus at increasing temperatures. Animals slowed down slightly in
their response above 18°C, and became significantly slower at 28°C
(N=15, *P<0.05, repeated measures ANOVA). The
percentage of animals not responding at all is shown in B: all animals righted
themselves between 12 and 24°C, no animal was able to turn at 30°C.
(C) Heart rate of not experimentally stimulated Cancer irroratus
increased with a Q10 of 2.2 between 12 and 26°C and
leveled off at 153±27 beats min–1 at 26°C before
decreasing again above 30°C (N=6). (D) Lactate concentration in
the heart tissue increased significantly above 26°C (N=6,
*P<0.05, ANOVA). The vertical dashed line indicates the
critical temperature (Tc), the vertical dotted line
indicates the pejus temperature (Tp).

AMPK and HSP70

AMPK activity (western blot for T172 phosphorylation) did not differ
between 12 and 18°C. AMPK activity started to increase above 18°C and
reached a maximum at 28°C [9.9(±2.3)-fold, P<0.05,
ANOVA; Fig. 4A]. The high
variability of the data is consistent with the high variability of the scope
for exercise of the individual animals (as shown by the reaction time data in
Fig. 3A,B). To test for a
discontinuity in the data, a Q-BASIC program to identify critical points
(Yeager and Ultsch, 1989) was
used. We identified two significantly different linear regressions
(y=1.21–5.26E–03x, R2=0.8659,
P<0.05; y=–12.76+0.75x,
R2=0.8067, P<0.05). The two regressions intersect
at 18.5°C. The increase in AMPK activity above 18°C coincides with the
decrease in reaction time (Fig.
3A). AMPK protein (Fig.
4B) and AMPKα mRNA levels
(Fig. 4C) showed the same small
decreases and increases, which remained statistically insignificant
(P>0.05, ANOVA) during the progressive temperature increase.

The increase of AMPK activity above 18°C occurred before any
significant changes in HSP70 protein or mRNA levels were detected
(Fig. 4D–F). A
non-significant upward trend of inducible HSP70 protein and HSP70 mRNA can be
seen at 28°C. This might indicate the onset of the heat shock response,
which would coincide with the inability of most animals to respond after being
turned on their backs (Fig. 3B)
and the onset of anaerobic metabolism (Fig.
3D).

Effect of constant temperature stress

The fast, progressive temperature increase described above elicits a quick
and immediate cellular response to thermal stress, as shown by the rapid
phosphorylation of AMPK. To test whether AMPK and HSP70 are affected
differentially during prolonged thermal stress, we exposed the animals for
various periods of time to the sublethal temperature of 26°C. Exposure to
26°C for up to 6 h led to a constantly high heart rate above 150 beats
min–1 (Fig.
5A). The lactate concentration in the heart peaked after 4 h at
23.4±8.5 nmol g–1 protein and remained above control
levels (15.3±0.8 nmol g–1 protein) after 6 h
(Fig. 5B). AMPK activity
remained constant at the high level that was reached at 26°C
(Fig. 5C). AMPKα mRNA
levels increased continuously throughout exposure to 26°C, reaching
5.6±2.2 times more after 6 h at 26°C than in the 12°C controls,
but reached statistical significance after 4 and 6 h, only at the
P<0.1 level, ANOVA (Fig.
5D). HSP70 protein levels (total and inducible) remained
constantly low with no significant changes
(Fig. 5E,F). HSP70 mRNA levels
rose constantly throughout the 6 h, up to 6.8(±1.7)-fold above control
(Fig. 5G). Thus, after this
prolonged exposure to high temperatures, both AMPKα mRNA and HSP70 mRNA
increased. The HSP70 protein did not follow the same trend as HSP70 mRNA. Our
experimental protocol most likely did not account for the time lag of protein
synthesis from mRNA. However, this was not the intention of the experiment
and, with the AMPK activity rising before either HSP70 protein or mRNA
increased, was not followed further.

DISCUSSION

AMPK is the subject of active research in the medical community, which
focuses on AMPK functions in homoeothermic mammals in response to cellular
ischemia and metabolic stress. AMPK plays a role in obesity
(Kola et al., 2008), heart
failure (Hardie, 2008) and
diabetes (Koh et al., 2008).
In all of these diseases, the cellular ATP homeostasis is disturbed. Because
AMPK is an important regulator of ATP homeostasis, it plays a major role in
developing or actually treating the respective disease. However, little is
known about AMPK in invertebrates and the effects of temperature on AMPK
activity. As expected, we found a high sequence conservation for the AMPK
protein in crustaceans. The sequence conservation across diverse phyla
indicates an important function in cellular metabolism, and maintenance of
cellular ATP concentration is probably the most important form of homeostasis.
We have shown that AMPK is expressed in C. irroratus, that
temperature stress leads to AMPK activation, and that this activation occurs
well before the well-characterized heat shock response with HSP70.

It is likely that a deeper understanding of the physiological processes
involved in withstanding temperature stress will allow for predictions of the
potential impacts of temperature change on animals. Once stress markers have
been identified, they can be used to evaluate the stress level of the
respective animal. Because extreme temperatures lead to anaerobiosis in the
tissues, temperature tolerance and hypoxia tolerance are related. Therefore,
elucidating the cellular mechanisms involved enhances our understanding of how
common changes in the environment, such as temperature and oxygen
concentration, will affect marine crustaceans' survival and their geographical
distribution range. This is especially important in the context of global
climate change or increasing hypoxic benthic areas (e.g.
Diaz and Rosenberg, 2008).

Representative western blots and the respective quantification for
phosphorylated and therefore activated AMPK (p-AMPK), heat shock protein 70
(HSP70 inducible and total) and the loading control actin, for heart tissue of
Cancer irroratus at temperatures between 12 and 28 or 30°C during
a fast and progressive temperature increase. (A) AMPK activity remained
constant between 12 and 18°C, increased above 18°C and reached
significance at 26°C. Two significantly different linear regressions
(dashed lines, for equations see text) were fitted using a Q-BASIC program to
identify critical points (Yeager and
Ultsch, 1989). The two regressions intersect at 18.5°C. (B)
Total AMPKα protein remained constant during the fast progressive
temperature increase. (C) Total AMPKα mRNA remained constant during the
fast progressive temperature increase. (D) Total HSP70 protein did not show
any significant changes during the temperature stress. (E,F) Inducible HSP70
protein and mRNA did not show any significant changes during the temperature
stress. However, the slight increase at 28°C might indicate the onset of
the heat shock response. For all figures: error bars show ±1 s.e.m.,
N=4–6 per data point, *P<0.05
vs 12°C, ANOVA. The vertical dashed line indicates the critical
temperature (Tc), the vertical dotted line indicates the
pejus temperature (Tp).

We chose the rock crab, C. irroratus, as a model species because
it can easily be obtained and maintained. It is one of the three major decapod
species of economic importance in the Gulf of Maine
(Palma et al., 1999), and the
crab fishery is currently expanding, especially in Canada
(Gendron et al., 2001). A
decline in the rock crab population could also have a negative impact on the
lobster fisheries in the Gulf of Maine because rock crabs play an important
role in the diet of the American lobster, Homarus americanus
(Gendron et al., 2001). A
multitude of earlier studies have characterized the physiological response of
decapod crustaceans to temperature stress. The current study builds on the
wealth of existing knowledge and contributes to an enhanced understanding of
the cellular and molecular processes affected by temperature stress.
Preliminary data from an earlier study showed that hypoxia affects AMPK
activity (Pinz et al., 2005),
and long-term temperature stress affects AMPKγ mRNA expression
differentially in different tissues of C. irroratus
(Frederich et al., 2006). For
the current, more comprehensive study, we chose to focus on the heart only,
because the temperature-induced effects on heart rate are well described and
can easily be monitored by a heart rate sensor. We are aware that heart rate
is a sub-optimal measure of cardiac workload. However, for the purpose of this
study, the temperature-induced increase in heart rate indicates increased
performance and, therefore, increased energy demand of this organ.

As outlined in the Introduction, AMPK is phosphorylated and activated
through AMPKK by a change in the cellular free AMP concentration. The AMP
concentration does not change only in the context of temperature stress.
Potentially every stress that affects cellular energy metabolism and ATP
hydrolysis rates, such as exercise, hypoxia, salinity stress and many others,
will lead to changes in cellular AMP and, consequently, affect AMPK activity.
Preliminary data show that salinity stress affects AMPK mRNA expression and
AMPK activity in salmon, Salmo salar, as well as AMPK activity in the
green crab, Carcinus maenas (M.F. and J.A.J., unpublished
observations). Jibb and Richards (Jibb and
Richards, 2008) show in their recent study that 0.5 h of hypoxia
in goldfish leads to a 5.5-fold increase in AMPK activity in the liver.
Consequently, we expect further studies to show that AMPK, as a central
regulator of cellular energy metabolism, is involved in many kinds of stress
response. Furthermore, we expect a dual mechanism to achieve this regulation.
First, a fast and immediate response through AMPK activation by
phosphorylation of AMPKα at the T172 site. This provides increased AMPK
activity within seconds. Second, a slower but longer lasting response through
increased AMPK mRNA and consequently AMPK protein expression. This provides a
long-term adjustment to varying energy demand. Both mechanisms are supported
by our study. A third possibility is a differential expression of AMPK subunit
isoforms. Whether invertebrates express the same set of isoforms as mammals
(see Introduction) is currently not clear. We are aware of only one
invertebrate study that claims to demonstrate two different AMPKα
isoforms, in the brine shrimp, Artemia franciscana
(Zhu et al., 2007).

(A) Keeping Cancer irroratus for 6 h at 26°C led to a
constantly high heart rate of 160.9±11.9 beats min–1.
(B) Lactate in the heart peaked after 4 h and remained elevated at 6 h. (C)
AMPK remained activated throughout the temperature stress. (D) AMPKα
mRNA levels showed an upward trend over the 6 h but reached statistical
significance only at the P<0.1 level (ANOVA). (E–G) HSP70
protein (total and inducible) remained constant while HSP70 mRNA levels
increased slowly and reached significance at 2, 4 and 6 h. For all figures,
the very first data point in each graph represents the value at 12°C for
each respective parameter before the temperature increase.
N=5–6 per data point, *P<0.05
vs 12°C, ANOVA.

Water temperatures in Casco Bay, where the crabs of this study were caught
(buoy GoMOOS CO2 at a depth of 2 m, black, and 20 m, grey). Adapted from
Shelford's law of tolerance and the adaptation by Frederich and Pörtner
(Frederich and Pörtner,
2000); we indicate the optimum range with a maximum scope for
exercise, limited by an upper pejus temperature, Tp. When
the animals are exposed to temperatures above Tp they
enter the pejus range with a limited scope for exercise and AMPK activity
increases to ensure an adequate cellular ATP concentration. Further
temperature increase leads to the critical temperature,
Tc, indicated by the onset of anaerobic metabolism,
lactate accumulation and HSP70 expression. Survival time in this pessimum
range is limited. Therefore, the first measured marker for cellular stress
through temperature is increased AMPK activity. For details see text.

AMPK and threshold temperatures

The activation of AMPK through heat stress can be viewed in the broader
theoretical framework of Shelford's law of tolerance
(Shelford, 1931) and critical
temperatures. Shelford describes an optimum and a pessimum range for animals
(and plants) for each respective environmental parameter. The ability to
survive is obviously at a maximum in the optimum range and gradually decreases
towards the upper and lower pessimum range. The following describes how
physiological parameters can identify thresholds between those ranges and how
AMPK activity can be used in a new way to characterize an additional range in
this model.

We detected an accumulation of lactate between 26 and 28°C in C.
irroratus, concomitant with attaining the maximum heart rate. Inducible
HSP70 protein, measured by western blot, as well as HSP70 mRNA, measured by
quantitative real-time PCR, showed a small but statistically insignificant
upward trend at this threshold. AMPK activity, however, was already well
elevated at Tc, with the increase commencing between 18
and 20°C. The response time after experimental stimulation increased
significantly above 26°C and coincided with lactate accumulation, maximum
heart rate, AMPK activity and an upward trend in HSP. The critical temperature
is therefore likely to occur between 26 and 28°C.

The threshold for the onset of increased AMPK activity coincides with the
temperature at which the response time first slowed down (18°C). This
initial slowing of response time was not statistically significant. However,
it might be biologically significant for a crab in its environment because the
ability to escape from a predator is crucial for survival. With a high
variability among individuals, some animals will be affected by heat stress
earlier and will be more vulnerable, as reflected in the higher standard error
in response time as well as AMPK activity. The onset of increased AMPK
activity coincides with the average maximum summer temperature in the area
where the animals were caught as well. Records of the Gulf of Maine Ocean
Observing System
(www.gomoos.org)
from 2002 to 2007 for Casco Bay (buoy CO2) at depths of 2 and 20 m show daily
average temperatures peaking in July at approximately 19°C and hourly
maximum temperatures of 20°C (Fig.
6). Animals used in the experiments were caught between late June
and early September and experiments were performed in the fall. The animals
therefore had a thermal history of maximum temperatures between 19 and
20°C. This is very close to the observed onset of AMPK activity between 18
and 20°C.

In an earlier study (Frederich and
Pörtner, 2000), we identified critical temperatures in the
spider crab, Maja squinado, and also an earlier threshold that we
called `pejus temperature, Tp' (pejus; latin for `getting
worse'). The upper and lower Tp encompass the range of
maximum performance and coincide with the normal habitat temperature. Animals
are exposed to temperatures in the pejus range, between Tp
and Tc, only occasionally. A recent study by Pörtner
and Knust (Pörtner and Knust,
2007) shows that eelpout in the North Sea are exposed briefly
every summer to temperatures above their pejus temperature, but rarely to
temperatures above their critical temperature. However, a reduction in growth
rate and relative abundance was clearly correlated to temperatures in the
pejus range, between Tp and Tc.
Therefore, the pejus temperature represents an important threshold that
describes the upper limit of regular function for an animal.
Fig. 6 summarizes the optimum,
pejus and pessimum range, as well as Tp,
Tc and the respective cellular processes described in the
present study. Habitat temperature data indicate why the cellular processes at
the Tp are more often relevant for the animals' survival
than the processes at Tc. Specifically, processes within
the pejus range, such as AMPK activity and the subsequent increased ATP
synthesis and reduced ATP use in anabolic pathways, help animals to save
ATP.

The actual value for Tp in C. irroratus, here
shown to be between 18 and 20°C, is likely to change with temperature
adaptation. During the winter, when ambient water temperatures are around
3°C, C. irroratus cannot survive much longer than 24 h at
23°C (M.F., personal observation). While the actual temperatures for
Tc and Tp shift with seasons and
probably vary among populations, depending on specific thermal conditions, the
mechanism is likely to remain the same. Animals are fairly tolerant to
temperature variations within their range of optimum performance between the
upper and the lower Tp. At the upper pejus temperature,
coinciding with the average seasonal maximum temperature, AMPK activity
increases to maintain an adequately high cellular ATP concentration. When
animals are exposed to much higher temperatures, anaerobic metabolism sets in
and survival is limited to a very short period of time. When exposed to
temperatures between Tp and Tc for
longer periods of time (in this study 6 h) the traditional heat shock response
starts, as shown by the increase in HSP70 mRNA after several hours at 26°C
(Fig. 5G).

Conclusion

In conclusion, to our knowledge this is the first study that specifically
investigates the effects of heat stress on AMPK activity in any animal. We
have shown that a fast, progressive temperature increase activates AMPK well
before the heat shock response can be observed via HSP70. The
temperature-related increase in AMPK activity coincides with a decrease in
responsiveness and therefore might be part of a mechanism that has significant
implications for the survival of the animals in their environment. We suggest
that the AMPK cascade represents a cellular mechanism to conserve ATP, which
allows the animals to endure short exposures to temperatures above the average
maximum temperature of their habitat. AMPK therefore, may be a potential early
cellular marker for heat stress in an animal.

LIST OF ABBREVIATIONS

AMPK

AMP-activated protein kinase

AMPKK

AMP-activated protein kinase kinase

HSP70

heat shock protein 70

Tc

critical temperature

Tp

pejus temperature

FOOTNOTES

Supported by a New Investigator Award from the Maren
Foundation at MDIBL, a Career Enhancement Award from the
American Physiological Society, NSF
IOB-0640478 to M.F., and a summer INBRE
fellowship to M.R.O. The authors thank David Towle and Chris
Smith from MDIBL for help with the quantitative real-time PCR methods and
sequencing, Elizabeth Mitchell and Christina Bucicchia for technical
assistance, Phil Yund for his help with the manuscript, and lobster boat
captain Roger Collard for generously providing the animals. This is
contribution number 18 from UNE's Marine Science Center.

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